A variety of specimens (e.g., clinical, environmental, food, processing equipment) are routinely tested to determine whether they contain microorganisms. Clinical samples such as urine, sputum, wound exudate, and the like, may have come in contact with skin or mucous membranes and can contain a variety of microorganisms. Some of the microorganisms in clinical samples are considered to be normal inhabitants of the skin or mucous membrane environment. Other microorganisms are considered pathogenic and can be associated with infectious disease processes.
Typically, one of the first steps in evaluating a specimen for viable pathogenic microorganisms is to grow the microorganisms to obtain large enough numbers to perform subsequent genetic, immunological, biochemical, and/or microscopic analyses. Often, the first step includes separating the individual microorganisms by spreading (or “streaking”) the sample onto a semisolid agar growth medium. This process results in the formation of individual, isolated colonies that can be used for subsequent testing.
Optionally, the agar growth medium on which the sample is spread can contain a differential reagent. Differential reagents can help with the classification and/or identification of the microorganisms growing on the medium. For clinical specimens, red blood cells (e.g., from a sheep or a horse) are often used as a differential reagent. Additionally, it is known in the art that components of blood (e.g., vitamins) can promote the growth of fastidious microorganisms. Certain pathogenic microorganisms, such as Streptococcus pyogenes and Staphylococcus aureus, secrete hemolysins that diffuse through the medium and lyse red blood cells, producing a distinctive zone around the colony. The hemolytic reactions can help with the early detection and identification of pathogenic microorganisms.
Production of differential semisolid medium containing red blood cells is a tedious process. The red blood cells are quite sensitive to thermal and osmotic shock, either of which can result in spontaneous lysis of the cells. The dehydrated agar is typically melted in water while mixing in a nutrient base. The mixture is subsequently sterilized by autoclaving and the temperature is lowered to a point where it is high enough to keep the agar molten but low enough to prevent the thermal lysis of the red blood cells. The mixture is then poured into containers, such as petri dishes. The agar is generally allowed to solidify before inoculating it with a sample. If the agar is not used immediately after it solidifies, typically it must be stored at refrigerator temperature and must used within several weeks because the agar begins to dehydrate and/or the red blood cells begin to spontaneously break down. If it is stored in a refrigerator, typically the agar medium is warmed to room temperature before use.
There exists a need for more convenient methods to detect hemolysin-producing microorganisms.